UC Berkeley

Here we present a microfluidic culture platform for enhancing single stem cell survival. Traditional plate culture is inadequate for large scale single cell studies because of (1) less than 40% single stem cell survival leading to (2) possible inaccuracies in the biostatistics in single cell studies from lack of sufficient data. This platform mitigates these issues by doubling overall survival rates as well as increasing the available data points by two orders of magnitude, both factors which improve statistical certainty. The platform is fabricated using widely accepted biocompatible polymers and incorporates the novel integration of an enclosed microwell array with an upstream linear gradient generator. The device can selectively trap single cells, tightly control intercell spacing, and change the chemical microenvironment around the stem cells in real-time. In this dissertation, we first characterize the performance of the platform in improving single cell survivability. Subsequently, the system was tested using two difference stem cell types: mouse embryonic stem cells (mES) and induced pluripotent stem cells (iPS). Using this platform we are able to highlight the thresholds of leukemia inhibitory factor concentrations which lead to metastable gene expression of Nanog, a key stem cell pluripotency regulator in mES cells. The enhanced survival of single cells has also enabled the statistically significant observation of Nanog+, non-proliferative, single mES cells which are previously unreported in literature.